Process for performing polynucleotide separations

ABSTRACT

The invention recognizes the deleterious effects of trace, and even undetectable amounts of multivalent cations on the separation of mixtures of polynucleotides, especially double stranded polynucleotides, and provides an improved method for separating such mixtures on wide pore, non-polar separation media by eliminating multivalent cations from the all aspects of the separation process. This is accomplished by using components in the separation process which are materials which do not release metal cations. In addition, the use of cation capture resins and other methods to remove residual traces of multivalent cations from eluting solvents, sample solutions, separation media, and system components is described. It is also important to remove any traces or organic contaminants from solvents solutions and system parts. Taking similar steps to remove residual traces of multivalent cations and organic impurities from the separation process, the invention may also be used in a batch process to separate mixtures of polynucleotide fragments.

RELATIONSHIP TO COPENDING APPLICATIONS

This is a division of Ser. No. 09/081,039, filed May 18, 1998, now U.S.Pat. No. 5,972,222, which, in turn, is a continuation-in-partapplication of Ser. No. 08/748,376 filed Nov. 13, 1996, now U.S. Pat.No. 5,772,889. This application is filed under 35 - U.S.C. §111(a) andclaims priority from commonly assigned provisional applications Ser. No.60/049,123 filed Jun. 10, 1997; and Ser. No. 60/063,835 filed Oct. 30,1997 under 35 U.S.C. §111(b).

FIELD OF THE INVENTION

This invention is directed to the separation of polynucleotide fragmentsby liquid chromatography. More specifically, the invention is directedto a system and method, which enhances the chromatographic separation ofpolynucleotides on non-polar, wide pore separation media.

BACKGROUND OF THE INVENTION

Separation of polynucleotide mixtures is a focus of scientific interest,and numerous researchers have been attempting to achieve technicalimprovements in various aspects of polynucleotide separation. Anionexchange separation and reverse phase ion pair chromatography are amongthe most frequently used methods for separating polynucleotide mixtures.

Samples containing mixtures of polynucleotides can result from totalsynthesis of polynucleotides, cleavage of DNA with restrictionendonucleases or RNA, as well as polynucleotide samples which have beenmultiplied or amplified using polymerase chain reaction (PCR) techniquesor other amplifying techniques.

Previous work has focused on developing rapid, high resolutionseparations, developing separations based on the size of thepolynucleotide fragment rather than the base sequence of the fragment,and on developing the ability to collect separated pure fractions ofpolynucleotides.

W. Bloch (European patent publication No. EP 0 507 591 A2) demonstratedthat, to a certain extent, length-relevant separation of polynucleotidefragments was possible on nonporous anion exchanger separation mediausing eluting solvents containing tetramethylammonium chloride (TMAC).Y. Ohimya et al. (Anal Biochem., 189:126-130 (1990)) disclosed a methodfor separating polynucleotide fragments on anion exchange materialcarrying trimethylammonium groups. Anion exchangers withdiethylaminoethyl groups were used by Y. Kato et al. to separatepolynucleotide fragments (J. Chromatogr., 478:264 (1989)).

U.S. Pat. No. 5,585,236 (1996) to Bonn et al. describes a method forseparating polynucleotides using what was characterized as reverse phaseion pair chromatography (RPIPC) utilizing columns filled with non-polar,nonporous polymeric beads. High resolution, rapid separations wereachieved using an ion pairing agent (triethylammonium acetate), andacetonitrile/water eluting solvent gradient. This work is importantbecause it is the first example of a size dependent, sequenceindependent chromatographic separation of double-strandedpolynucleotides by Matched Ion Polynucleotide Chromatography (MIPC).Such separations are comparable to those effected by gelelectrophoresis, which is currently the technology most widely used forpolynucleotide separations. Bonn's work makes it possible to automateseparations of polynucleotides based on their size alone. This methoddiffers from traditional reverse phase processes. Therefore, the termMatched Ion Polynucleotide Chromatography (MIPC) has been applied to theBonn process to distinguish it from previously known reverse phaseprocesses.

The invention of parent application Ser. No. 08/748,376 is based on thediscovery that trace levels of multivalent metal ions, even when presentbelow the limits of detection, interfere with the MIPC separationprocess. Special steps to prevent, remove or complex any tracemultivalent ions result in enhanced separation of polynucleotides andlower the detection threshold. The inventions of provisionalapplications Ser. No. 60/049,123 filed Jun. 10, 1997; and Ser. No.60/063,835 filed Oct. 30, 1997 under 35 U.S.C. §111(b) are based on thediscovery that nitric acid passivated stainless steel, titanium, andPEEK (polyetherether ketone) surfaces were, contrary to popular belief,sources of multivalent metal ion contamination in the MIPC process. Thedeleterious effect of multivalent metal cations on polynucleotideseparations as observed herein has not been previously reported. Webelieve that all chromatographic processes which are capable ofseparating polynucleotides on non-polar, wide pore separation media areimpaired by the interference of multivalent metal ions.

SUMMARY OF THE INVENTION

Therefore, the invention provides an improved method for separating amixture of polynucleotide fragments wherein multivalent cations areeliminated from the all aspects of the separation process. The methodcomprises applying a solution of said fragments and counterion agent toa column containing separation media having a non-polar surface, whereinsaid separation media have a pore size greater than 30 Angstroms and anaverage diameter of 1-100 microns. Separation of said fragments isaccomplished by eluting said fragments with an eluting solvent gradientof increasing organic component concentration containing a counterionagent. Surfaces which are contacted by the solution of the fragments andthe eluting solvent are materials which do not release multivalent metalcations therefrom, said materials having been washed to remove traces oforganic contaminants therefrom. The method further comprises contactingthe solution of said fragments and the eluting solvent with amultivalent cation capture resin to remove any multivalent cationstherein before entering the column.

In a preferred embodiment of the invention, the separation media havebeen treated to remove residual traces of multivalent cations from thesurfaces therefrom.

An optimum embodiment of the invention comprises contacting the solutionof said fragments and eluting solvent with a multivalent cation captureresin before entering the column, treating the separation media toremove residual traces of multivalent cations from the surfacestherefrom, and ensuring that surfaces which are contacted by thesolution of the fragments and the eluting solvent are materials which donot release multivalent metal cations therefrom and cleaning saidsurfaces to remove any traces of organic contaminants therefrom.

In one embodiment, the polynucleotide fragments are double stranded,having more than 5 base pairs. Such fragments are separated by size orby polarity.

In another embodiment of the invention, the polynucleotide fragments aresingle stranded having 2 or more nucleotides. Such fragments areseparated by size and by polarity.

The separation media are organic polymer, or an inorganic substrateselected from the group consisting of inorganic substrates, silica,zirconia, and alumina. The inorganic substrates support a non-polarmaterial on their surface. Said non-polar material may be organicpolymer or long chain, C1 to C24 hydrocarbon groups bound to theinorganic substrate, wherein residual polar groups of the substrate areend capped with trimethylsilyl chloride or hexamethyldisilazane.

In a preferred embodiment, surfaces which are contacted by the solutionof polynucleotide fragments and eluting solvent are titanium, coatedstainless steel, organic polymer or combinations thereof. Removal oftraces of residual multivalent metal cations from the separation processis further ensured by treating said surfaces with a solution comprisingaqueous acid and chelating agent, by adding a chelating agent to thesolution of polynucleotide mixture and eluting solvent, and by treatingthe eluting solvent to remove oxygen therefrom.

In one embodiment, the improved method for separating said mixture ofpolynucleotides comprises Matched Ion Polynucleotide Chromatography.

The improved method of the invention may also be practiced as a batchprocess for separating polynucleotide fragments having a selected sizefrom a mixture of polynucleotide fragments including fragments of saidselected size. The batch process method of the invention comprisesapplying a solution of said polynucleotide fragments and a counterionagent to non-polar separation media having a non-polar surface, whereinsaid separation media have a pore size greater than 30 Angstroms and anaverage diameter of 1-100 microns. The method further comprisescontacting the separation media with a first eluting solvent andcounterion agent, the first eluting solvent having a concentration oforganic component sufficient to release from the separation media allpolynucleotide fragments having a size smaller than the selected sizeand removing the first eluting solvent from the separation media. Theselected size fragments are obtained by contacting the separation mediawith a second eluting solvent having a concentration of organiccomponent sufficient to release from the separation media thepolynucleotide fragments having the selected size and removing thesecond eluting solvent from the separation media. Preferably, surfaceswhich are contacted by the solution of polynucleotide fragments and theeluting solvent are material which does not release multivalent metalcations therefrom.

Following removal of the first eluting solvent, the separation media arerinsed with fresh first eluting solvent to remove residual releasedpolynucleotide fragments therefrom. In a similar manner, followingremoval of the second eluting solvent the separation media are rinsedwith fresh second eluting solvent to remove residual releasedpolynucleotide fragments of selected size therefrom.

A preferred embodiment of the invention comprises contacting thesolution of polynucleotide mixture and eluting solvent with amultivalent cation capture resin before contacting the separation media.In another preferred embodiment the method comprises treating theseparation media to remove residual traces of multivalent cationstherefrom. Optimally the separation media have been treated to removeresidual traces of multivalent cations therefrom and the solution ofpolynucleotide mixture and eluting solvent have been contacted with amultivalent cation capture resin before contacting the separation media.Said separation media are contained in a column, a web, a membrane, orcontainer.

The batch process can be used to separate mixtures of double strandedpolynucleotides or single stranded polynucleotides.

The separation media are organic polymer, or an inorganic substrateselected from the group consisting of inorganic substrates, silica,zirconia, and alumina. The inorganic substrates support a non-polarmaterial on their surface. Said non-polar material may be organicpolymer or long chain, C1 to C24 hydrocarbon groups bound to theinorganic substrate, wherein residual polar groups of the substrate areend capped with trimethylsilyl chloride or hexamethyldisilazane.

The surfaces contacted by the solution of polynucleotide fragments andeluting solvent are, preferably, comprised of material selected from thegroup consisting of titanium, coated stainless steel, and organicpolymer, or combinations thereof. Removal of traces of residualmultivalent metal cations from the separation process is further ensuredby treating said surfaces with a solution comprising aqueous acid andchelating agent, by adding a chelating agent to the solution ofpolynucleotide mixture and eluting solvent, and by treating the elutingsolvent to remove oxygen therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a guard disk having a one-piece annular ring.

FIG. 2 is an exploded view of a guard disk having a two-piece annularring and containing three pads of guard disk material (i.e., a layer orpad of multivalent cation capture resin which has been incorporated intoa fabric or membrane).

FIG. 3 shows an assembled view of the guard disk of FIG. 2.

FIG. 4 shows placement of a chelating guard column and chelating guarddisk in a liquid chromatographic system for polynucleotide separation.

FIG. 5 shows placement of a chelating guard disk positioned between achromatographic separation column and a column top, where the guard diskis in direct contact with a titanium frit at the top portion of theseparation column.

FIG. 6 shows the chromatography of a 500 base pair DNA fragment on PRX-1(Sarasep, San Jose, Calif.) non-polar, wide pore polymer separationmedia which have been washed (see Example 1) to remove multivalent metalcations thereform.

FIG. 7 shows the effect on the chromatography result shown in FIG. 6 henCr(III) cations were added to the column before sample injection.

FIG. 8 shows the effect on the chromatography result of FIG. 7 when aCr(III) contaminated column was treated with EDTA prior to sampleinjection.

FIG. 9 shows the chromatography of a 500 base pair DNA fragment onINERTSIL (MetaChem, Torrance, Calif.) C-18 non-polar, wide pore silicaseparation media which have been washed with EDTA to remove multivalentmetal cations thereform.

FIG. 10 shows the effect on the chromatography result shown in FIG. 9when Cr(III) cations were added to the column before sample injection.

FIG. 11 shows the effect on the chromatography result of FIG. 10 when aCr(III) contaminated column was treated with EDTA prior to sampleinjection.

FIG. 12 shows the chromatography of a 20 mer single stranded DNAfragment standard (Seq 2A, CTGen, Milpitas, Calif.) on INERTSIL(MetaChem, Torrance, Calif.) C-18 non-polar, wide pore silica separationmedia which have been washed with EDTA to remove multivalent metalcations thereform.

FIG. 13 shows the effect on the chromatography result shown in FIG. 12when Cr(III) cations were added to the column before sample injection.

FIG. 14 shows the chromatographic separation at 56° C. of a 209 basepair DNA standard 4 component heteroduplex and homoduplex mixture on afreshly packed, untreated DNASep column (Transgenomic, Inc., San Jose,Calif.).

FIG. 15 shows the effect on the chromatography result shown in FIG. 14when the column was treated with EDTA prior sample injection.

DETAILED DESCRIPTION OF THE INVENTION

The term polynucleotide, as used herein, is defined as a linear polymercontaining an indefinite number of nucleotides, linked from one ribose(or deoxyribose) to another via phosphate residues. The presentinvention can be used in the separation of RNA or of double or singlestranded DNA. For purposes of simplifying the description of theinvention and not by way of limitation, the separation of doublestranded DNA will be described hereinafter, it being understood that allpolynucleotides are intended to be included within the scope of thisinvention.

The present invention is an improved chromatographic and batch processmethod for separating mixtures of polynucleotide fragments on wide porenon-polar separation media. The improvement comprises ensuring theremoval of all traces of residual multivalent metal cations from thesample mixture, the eluting solvent as well as components of thechromatographic or batch process equipment which contact said samplemixture and eluting solvent. The inventor's observation anddemonstration that even traces of multivalent metal cations below thelevel of detection, degrade polynucleotide separations on wide pore,non-polar separation media and that removal of said cations results inimproved separation efficiency and column life is both surprising andnovel.

The system used to implement the method of the invention comprises aliquid chromatographic system. The liquid chromatography systemcomprises a column containing a separation bed of non-polar, wide poreseparation media held in the column between porous frits positioned ateach end of the column. Other components of the liquid chromatographysystem include an injection valve and one or more eluting solvent supplymeans. Eluting solvent supply means is (are) connected to the injectionvalve, and the injection valve is connected to the inlet of thechromatographic separation column, by means of conduit (e.g., tubing),as illustrated in FIG. 5.

The chromatography system components mentioned hereinabove andvariations thereof are well known in the chromatography art anddescribed in detail in references cited hereinbelow.

In a preferred embodiment of the invention, the mixture ofpolynucleotide fragments is separated by Matched Ion PolynucleotideChromatography (MIPC). The term "Matched Ion PolynucleotideChromatography" as used herein is defined as a process for separatingsingle and double stranded polynucleotides using non-polar, wide poreseparation media, wherein the process uses a counterion agent, and anorganic solvent to release the polynucleotides from the separationmedia.

The pores of the separation media may be contiguous, i.e., extend fromone surface of the media to another surface of the media. The pores ofthe separation media may also be non-contiguous, i.e., extend into themedia at one point on the surface but not through to another point onthe surface.

The non-polar, wide pore separation media can be an inorganic substrate,including silica, zirconia, alumina, or other material; or can bepolymeric, including crosslinked resins of polystyrene, polyacrylates,polyethylene, or other organic polymeric material. The non-polar, widepore separation media can also be a "rod column" or "monolith column".Such columns contain silica or polymer separation media which have beenformed inside the column as a continuous structure which has throughpores or interstitial spaces which allow eluting solvent and analyte topass through. The only requirement for the non-polar, wide poreseparation media is that they must have a surface that is eitherintrinsically hydrophobic or be bonded with a material that forms asurface having sufficient hydrophobicity to interact with a counterionagent.

As used herein, the term "non-polar, wide pore separation medium" isdefined to denote any material which has surface pores having a diameterthat is greater than 30 Angstroms or the approximate size and shape ofthe smallest polynucleotide fragment in the separation in the solventmedium used therein or greater, and is capable of separatingpolynucleotide fragments. Although the non-polar, wide pore separationmedium may be any shape, those comprising alkylated wide pore polymerbeads bead having an average diameter of 1-100 microns are preferred.Such particles are described in the references cited hereinbelow.

Regardless of the source or composition of the non-polar, wide porereverse phase separation media described above, special precautions aretaken to ensure that they are substantially free of multivalent cationcontaminants. For example, the separation media are washed with acidfollowed by methanol to ensure removal of residual multivalent cationcontaminants. The separation media can also be washed with EDTA or otherchelating agent.

The concepts, materials, systems and methods related to chromatographyon non-polar, wide pore separation media are well known and aredescribed in detail in the following references: Chromatography Today,by Colin F. Poole and Salwa K. Pool, Elsevier (1991); Introduction toModern Liquid Chromatography, L. R. Snyder and J. J. Kirland, J. Wileyand Sons, Inc. (1979). These references and references contained thereinare incorporated in their entirety herein.

Non-polar, wide pore separation media and their use for the separationof polynucleotide mixtures are well known in the art and arecommercially available, e.g., Hamilton HPLC Application Handbook,(1993), Hamilton Company, Inc., 4970 Energy Way, Reno, Nev. 89502. This,and references contained therein, are incorporated in their entiretyherein. Another reference, which is incorporated in its entirety herein,describing polynucleotide separations on non-polar, wide pore separationmedia is Chromatography, 5th edition, Part B, edited by E. Heftmann,Elsevier (1992). Separation of tRNA and DNA fragment mixtures onnon-polar, wide pore silica particles is described by R. Bischoff and L.W. McLaughlin, Analytical Biochemistry, 155, 526-533 (1985) and S.Eriksson, et. al., J. Chromatography, 359, 265-274 (1986).

Monolith or rod columns are commercially avialable form Merck & Co(Darmstadt, Germany) and described in the following references: U.S.Pat. No. 5453185 to J. M. J. Frechet and F. Svec; M. Petro, et. al.,Analytical Chemistry, 68, 315-321 (1996). The references cited above andthe references contained therein are incorporated in their entiretyherein.

The components of the liquid chromatography system have surfaces (i.e.,"process solution contacting surfaces") which contact process solutionsheld within the components (e.g., the eluting solvent supply means) orflowing through the components (e.g., the porous frits, chromatographiccolumn, injection valve, and conduits). The term "process solution" asused herein refers to any solution (such as the polynucleotide mixturesolution and the eluting solvent) which is contained within or flowsthrough any component of the liquid chromatography system during thechromatographic process. The term "process solution contacting surface"refers to any surface of a liquid chromatography system which contactssaid process solutions.

The process solution contacting surfaces of the porous frits on eitherend of the separation column must be made of material which does notrelease multivalent cations into solutions flowing through the column,or collect said cations from other sources. The material is preferablytitanium, coated stainless steel, or organic polymer, or combinationsthereof, but is most preferably acid treated titanium as describedhereinbelow. The term "coated stainless steel" as used herein refers tostainless steel that has been coated so that it does not release, or isprevented from releasing, multivalent cations. A non-limiting example ofa coating material is polytetrafluoroethylene (i.e., Teflon®). "Coatedstainless steel" as used herein also refers to stainless steel that hasbeen pre-treated with an agent such as EDTA or phosphoric acid whichforms coordination complexes with multivalent metal ions.

"Passivated stainless steel" as used herein refers to stainless steelthat has been treated with an agent that removes oxidized metals andalso metals that are easily oxidized such as iron. The most commonpassivating agent for stainless steel is nitric acid. Nitric acid willremoved any oxidized metals, but will also remove iron that is locatedon the surface of the metal, leaving other metals such as chromium andnickel. Some chelating agents can coat and passivate. EDTA will firstcoat oxidized metals especially colloidal iron oxide particles. Astreatment continues, the EDTA will bind and dissolve the iron oxide.However, as individual iron molecules leave the particle, otherchelating molecules must coat the newly exposed surfaces for the surfaceto remain suitable for polynucleotide separations. A chelating agentdoes not passivate in the sense that it will only coat metal ions forwhich it is specific and will not dissolve non-oxidized metals. However,a chelating agent may, eventually, dissolve oxidized metals.

The chelating agents used depend upon the type of ion contaminationwhich is present. For example, Tiron chelating agent is selective fortitanium and iron oxides. EDTA is selective for most metal oxides at pH7. Other chelating agents include cupferron, 8 hydroxyquinoline, oxine,and various iminodiacetic acid derivatives. If the chelating agents areto be used as passivating reagents as well as coating reagents, then itis important that the metal ion chelate complex, for example, EDTA-metalion complex, is soluble in the fluid. Chelating agents that forminsoluble complexes, for example 8-hydroxyquinoline, perform coatingfunctions only.

Without wishing to be bound by theory, it is believed that oxidized andpositively charged metals, such as oxides of iron on the surface ofstainless steel can trap negatively charged molecules such as DNAleading to degradation of the chromatographic separation, and that thepre-treatment masks or shields these surface charges. EDTA can be added,for example, in an amount sufficient to shield any surface sites whichwould interfere with the chromatographic separation. In one embodiment,a solution of a metal chelating agent such as EDTA can be applied in abatch process to coat the surface, for example by a single injection ofEDTA solution into the HPLC system. In another embodiment, EDTA isincluded as an additive in the eluting solvent in an amount sufficientto complex the metal ions present.

Other components of the liquid chromatography system are preferablytitanium, coated stainless steel, or organic polymer such aspolyetherether ketone (PEEK) or polyethylene. The preferred systemtubing (i.e., conduit) is titanium, PEEK, or other polymeric material,with an inner diameter of 0.007". The preferred eluting solvent inletfilters are composed of non-polar, porous, non-stainless steel material,which can be PEEK, polyethylene, or other polymeric material. Thepreferred solvent pump is also made of a non-stainless steel material;the pump heads, check valves, and solvent filters are preferablytitanium, PEEK, or other polymeric material. The preferred means forremoving oxygen from the eluting solvent is an inline degasser placedprior to the pump inlet. The sample injection valve is also preferablytitanium, PEEK, or other polymeric material. A standard detector andeluting solvent reservoirs can be used, with no modifications necessary.

Materials such as titanium, PEEK and other organic polymers such aspolyethylene, have been generally considered to be inert and preferredfor the chromatographic separation of biological molecules. We havediscovered that these materials, while inert for the prior artprocesses, can be a source of contaminants which interfere with thechromatographic separation of polynucleotides on non-polar, wide poreseparation media. We have also observed that the interference withseparation of polynucleotides by these materials becomes more apparentduring separations carried out at elevated temperatures, e.g. 56° C. ascompared to 50° C.

In a preferred embodiment of the present invention, all of the processsolution-contacting surfaces are subjected to a multivalent cationremoval treatment to remove any potential source of multivalent cationcontamination. These surfaces include the column inner surface, porousfrits, conduits, eluting solvent supply system, injector valves, mixers,pump heads, and fittings. A non-limiting example of a multivalent cationremoval treatment is an acid wash treatment. This wash treatment caninclude flushing or soaking and can include sonication. An example of anacid wash treatment is sonication of a PEEK or titanium frit in thepresence of aqueous nitric acid solution, followed by sonication inwater until a neutral pH is achieved. Other treatments includecontacting the surfaces with chelating agents such as EDTA,pyrophosphoric acid, or phosphoric acid (e.g. 30% by weight phosphoricacid).

PEEK and titanium can be treated with dilute acids including nitric andhydrochloric acids. PEEK is not compatible with concentrated sulfuric orconcentrated nitric acids. Titanium is not compatible with concentratedhot hydrochloric acid. Treatment with a chelating agent can be performedbefore, but preferably after treatment with an acid. 20 mM tetrasodiumEDTA is a preferred chelating agent treatment.

The preferred treatment for titanium frits is sonication for 10 minuteswith cold hydrochloric acid, sonication with water until neutral pH, 2hour sonication with 0.5 M tetrasodium EDTA, storage several days in 0.5M tetrasodium EDTA, sonication with water until neutral pH, and thenwashing with methanol, followed by drying. Preferred treatment for PEEKfrits is sonication for 15-30 minutes each with THF, concentratedhydrochloric acid, 20% nitric acid, sonication with water until neutralpH, and then washing with methanol, followed by drying. Although this isa preferred treatment method, the effectiveness of this treatment ofPEEK frits can depend on the vendor and lot of material treated. Thesuccess of the treatment also depends on the temperature of thepolynucleotide separation with higher column temperatures requiring themost complete removal of contamination. If the ionic contaminant isorganic, then organic solvents or a combination of organic solvents andacids can be used. Also, organic ionic contaminants can requiredetergents, soaps or surfactants for removal from the surface.

Nonionic contaminants such as greases and oils will also contaminate theseparation column, generally leading to poor peak shape, but dependingupon the size of the fragment. Nonionic organic contaminants such asoils will require detergents, soaps or surfactants to remove. Columntubing can be treated under sonication with Decalin (D5039, Sigma) toremove silicon greases and oils. Removal of colloidal metal oxides suchas colloidal iron oxide can require repeated or continuous treatment asthe surface of the particle is dissolved and new metal oxides areexposed.

The preferred embodiment of the liquid chromatography system of thepresent invention utilizes methods to minimize the exposure of allprocess solution contacting surfaces to oxygen. Dissolved oxygen withinthe eluting solvent, for example, can react with exposed metals on thesesurfaces to form oxides which will interfere with the chromatographicseparation.

The liquid chromatography system preferably employs a degassing methodfor essentially removing dissolved oxygen from the eluting solvent priorto contact with the rest of the chromatography system. Examples ofdegassing methods include sparging of the eluting solvent with an inertgas such as argon or helium, or filtering the eluting solvent undervacuum. A preferred method uses a vacuum type degasser which employsinline passage of the eluting solvent over one side of an oxygenpermeable membrane system where the other side is subjected to a vacuum.An example of a suitable four channel vacuum type degasser is Degaset™,Model 6324 (MetaChem Technologies, Torrance, Calif.).

In another embodiment of the invention, a stainless steel HPLC systemcan be used if a component for removing multivalent cations, hereinreferred to as a "multivalent cation capture resin," is also used. Amultivalent cation capture resin is preferably a cation exchange resinor chelating resin. Any suitable cation exchange resin or chelatingresin can be used. Preferred cation exchange and chelating resins aredescribed hereinbelow.

Cation exchange resins having an ion exchange moiety selected from thegroup consisting of iminodiacetate, nitriloacetate, acetylacetone,arsenazo, hydroxypyridinone, and 8-hydroxyquinoline groups areparticularly preferred. Cation exchange resins having hydroxypyridinonegroups are especially useful for removing iron from the system. Cationexchange resins having iminodiacetate groups are particularly preferredfor use in the present invention because of their wide availability inresin format.

A chelating (i.e., coordination binding) resin is an organic compoundwhich is capable of forming more than one non-covalent bond with ametal. Chelating resins include iminodiacetate and crown ethers. Crownethers are cyclic oligomers of ethylene oxide which are able to interactstrongly with alkali or alkaline earth cations and certain transitionmetal cations. A cavity in the center of the molecule is lined withoxygen atoms which hold cations by electrostatic attraction. Each etherhas a strong preference for cations whose ionic radius best fits thecavity.

The multivalent cation capture resin is preferably contained in a guardcolumn, guard cartridge, or guard disk. Guard columns and cartridges arefrequently used to protect liquid chromatography columns fromcontamination and are widely available. In their normal use, guardcolumns and cartridges typically contain packing material which issimilar to the stationary phase of the separation column. However, foruse in the present invention, the guard column or cartridge must containa multivalent cation capture resin. The guard disc or guard column mustcontain particles which trap the metal ions.

For use in the system of the present invention, the guard cartridge orcolumn should be sufficiently large to provide adequate cation capturecapacity, but must be small enough to allow effective gradient elutionto be used. A preferred guard cartridge has a void volume of less than 5mL, more preferably, less than 1 mL, so that the eluting solventgradient is not delayed by more than 5 minutes and, preferably, lessthan 1 minute. The preferred cartridge has a 10×3.2 mm bed volume.

Guard disks are described in detail in U.S. Pat. No. 5,338,448, which isincorporated herein by reference in its entirety. For use in the presentinvention, a guard disk comprises a layer or pad of a multivalent cationcapture resin which has been incorporated into a fabric or membrane sothat the resin is not separable from the guard disk under liquid flowconditions present during the performance of chromatographicseparations.

In its preferred form, the guard disk is circular, having a rigidannular outer ring or collar for easy handling. The annular ring can beconstructed of any suitable material which is inert to thechromatographic separation, such as inert conventional engineeringplastic. The only requirement for the material is that it must be inertto the eluting solvent and sample and have sufficient dimensionalstability. The rigid annular outer ring of the guard disk can comprise asingle rigid annular outer ring encircling a disk-shaped pad of guarddisk material. As used herein, the term "guard disk material" refers toa layer or pad of multivalent cation capture resin which has beenincorporated into a fabric or membrane.

As shown in FIG. 1, one or more pads of guard disk material 2 are placedin the rigid annular ring 4. For example, the fabric can be cut to acircular diameter which securely contacts the inner diameter surface ofthe annular ring. As the disk holder is tightened against the disk, thetop and bottom surfaces of the holder seal against the collar of theguard disk. Sealing pressure from the guard disk holder is, therefore,applied against the collar of the disk which prevents the material ofthe guard disk pad from being crushed.

Alternatively, the rigid annular outer ring can comprise two flangedrings, as shown in FIG. 2 and 3, an outer flanged ring 6 and an innerflanged ring 8, where the inner flanged ring is insertable within theflange of the outer ring, forming a press-fit two-piece collar aroundone or more pads of guard disk material 10. Preferably, the innerdiameter (a) of the inner flanged ring will have the same diameter asthe separation column bed.

In the two-piece annular ring embodiment shown in FIG. 3, one or morepads of guard disk material 10 having a diameter greater than the innerdiameter (b) of the outer flanged ring 6 are positioned within theflanges of the outer ring. The inner flanged ring 8 is then insertedinto the outer ring to form a press-fit two-piece annular ring in whichthe guard disk pad(s) is (are) frictionally held within the press-fitring or collar. Preferably, the inner diameter (b) of the outer flangedring and the inner diameter (a) of the inner flanged ring aresubstantially the same.

Alternatively, the rigid annular outer ring can be incorporated into theguard disk holder or chromatographic column cap. The annular ring is aflange that is part of one or both sides of the disk holder or thecolumn cap. In this case, the guard disk does not have an outer ring. Acircle of the guard disk sheet material is placed into the holder orcolumn cap. The flange in the holder column cap is annular so that, whenthe holder or column cap is tightened, the flange pinches or seals theouter annular portion of the guard disk. The center portion of the guarddisk not pinched is in a chamber or depression in the holder or cap.Fluid flows through the center portion, allowing the guard disk toretain particulate or strongly adsorbed material, but fluid cannot flowaround the disk or past the edges. The function of the guard disk isexactly the same as when the collar is part of the guard disk itself.However, in this case, the collar is part of the holder or column cap.

In a most preferred embodiment of the invention, a multivalent cationcapture resin contained in a guard column, guard cartridge, or guarddisk is placed upstream of the separation column. Most preferably, theguard column, cartridge, or disk containing the resin is placed upstreamof the sample injection valve. Although this is preferably a guard disk,a guard cartridge or column can be used as long as the dead volume ofthe cartridge or column is not excessive and an effective elutingsolvent gradient can be produced.

Optimally, a guard disk, column, or cartridge can be placed before theinjection valve and a second guard disk, column, or cartridge alsoplaced between the sample injection valve and the separation column. Incertain cases, the second guard disk (or cartridge or column) can beavoided if the contaminants are sufficiently cleaned by a guard columnplaced upstream of the injection valve, or if the contaminants areavoided through the use of non-metal or titanium components throughoutthe HPLC system.

Placement of a chelating guard column and chelating guard disk in aliquid chromatography system for polynucleotide separation isillustrated in FIG. 4. The eluting solvent reservoirs 12 contain elutingsolvent inlet filters 14 which are connected to the solvent pump 16 bysystem tubing 18. The solvent pump 16 is connected to a chelating column20 by system tubing 18. The chelating column 20 is connected to thesample injection valve 22 by system tubing 18. The sample injectionvalve has means for injecting a sample (not shown). The sample injectionvalve 22 is connected to a chelating guard disk 24 by system tubing 18.The chelating guard disk 24 is connected to the inlet (not shown) of theseparation column 26 by system tubing 18. Detector 28 is connected tothe separation column 26. As discussed above, the system tubing, elutingsolvent inlet filters, solvent pump, sample injection valve, andseparation column are preferably made of titanium, coated stainlesssteel, or organic polymer. The material is preferably treated so that itdoes not release multivalent cations. The treatment can includetreatment with nitric acid, phosphoric acid, pyrophosphoric acid, orchelating agents. In cases, where components of the HPLC do not releasemetal ion contaminants and are suitable for polynucleotide separationsin general and MIPC in particular, then use of the chelating cationexchange guard column or guard disc is not necessary.

In operation, eluting solvent from the eluting solvent reservoirs 12 ispumped through eluting solvent inlet filters 14 by solvent pump 16. Byway of system tubing 18, the eluting solvent stream flows throughchelating column 20, through sample injection valve 22, throughchelating guard disk 24, then into separation column 26. Detector 28 islocated downstream from separation column 26.

FIG. 5 illustrates a specific embodiment of the invention in which achelating guard disk is placed in direct contact with a titanium frit atthe top portion of a chromatographic separation column. Column top 30has conventional fittings for receiving eluting solvent and samplethrough inlet tubing 32. The column top or cap 30 is fitted and sealablyattached to column body 34 containing chromatographic bed 36 using aconventional fitting 38 (e.g., threaded) or any equivalent fittingcapable of tightly sealing the column top to the column body. The columntop 30 is adapted to receive the chelating guard disk 40 in a sealingcavity 42. In this embodiment, the guard disk 40 is in direct contactwith a titanium column frit 44, which is located at the upstream end ofthe column body 34 to prevent disturbance of the chromatographic bed 36when the column top 30 is removed to observe the guard disk.

In operation, solvent pump 46 pumps elution solvent to sample injectionvalve 48 into column top 30 through chelating guard disk 40 and thenthrough titanium frit 44 before entering chromatographic bed 36. Elutingsolvent pressure upstream from the guard disk is measured by pressuretransducer 50 which is electrically connected to a display device 52.

As discussed above, a chelating guard column, cartridge, or disk can beused in conjunction with a conventional, stainless steel liquidchromatography system, or with a system containing non-metal or titaniumcomponents in order to provide extra protection against ioniccontaminants. For additional column protection, an eluting solventcontaining 0.1 mM tetrasodium EDTA or other chelating solution can beused during the performance of polynucleotide separations.

In another aspect of the invention the non-polar, wide pore separationmedia have been washed to remove any traces of residual multivalentmetal cations from the surface thereof. Preferred washing solventscomprise tetrahydrofuran, hydrochloric acid, and water. An example of apreferred washing procedure is described in Example 1.

The methods of the invention comprise using the improved systemsdescribed above to separate mixtures of polynucleotide fragments,particularly double-stranded polynucleotide fragments. The methods ofthe present invention can be used to separate polynucleotide fragmentshaving up to about 1500 base pairs using non-polar, wide pore separationmedia under the chromatography conditions described herein.

The most preferred method of the invention comprises contacting asolution of a mixture of polynucleotide fragments containing acounterion agent with a multivalent metal cation capture resin, followedby application of said solution to a separation column containingnon-polar, wide pore separation media wherein said particles have beenwashed to remove any traces of residual multivalent cation therefrom.Optimally, the process solution contact surfaces of the system have beenpassivated, as described hereinabove, to remove multivalent metalcations therefrom. In an optimum configuration, a guard cartridge orguard column containing multivalent cation capture resin is placed atthe front of the column or in line between the eluting solvent reservoirand the solvent pump(s) to protect said column and the separation mediacontained therein from any traces of residual multivalent metal cationsin the eluting solvent. The polynucleotide fragments are separated byreleasing said fragments from the separation media using an elutingsolvent comprising an organic component, water, and a counterion agent.The separation of the polynucleotide components is based on the size orpolarity of the fragments. By way of example only, the fragments arereleased from the separation media in order of size by increasing theconcentration of organic component in the eluting solvent. Theconcentration of the organic component can be increased in stepwisefashion by means of a step gradient, or continuously, by means of acontinuous gradient.

The methods used to capture multivalent cations and prevent theirpresence in the chromatography system, are essential in order to achievehigh resolution separations of polynucleotides, especially doublestranded DNA, and also to greatly extend the useful life of theseparation media. Evidence demonstrating the detrimental effect ofmultivalent metal cation contamination on the chromatographic separationof both single stranded polynucleotide fragments and double strandedpolynucleotide fragments on non-polar, wide pore silica and polymerseparation media is presented in Examples 2-7 and FIGS. 6-15.

Example 2 describes a polynucleotide fragment separation on non-polar,wide pore organic polymer separation media using the optimized method ofthe invention compared to a deliberate contamination of the system withmultivalent metal cations. While a sharp peak is obtained using theoptimized method of the invention, the peak is completely absent whenthe chromatography system was deliberately contaminated with multivalentmetal cations.

Example 3 is identical to Example 2, except that non-polar, wide poresilica separation media were used in the chromatography.

In Example 4, a series of three separations of a 500 base pair DNAfragment was performed using non-polar, wide pore polymer separationmedia are described. In the first separation, the separation media waswashed to remove multivalent cations as described in Example 1 prior tosample injection, and a sharp peak was obtained as shown in FIG. 6.Deliberate contamination of this separation media with Cr (III) cationsresulted in a complete loss of the sample peak as shown in FIG. 7.Treatment of the separation particles with EDTA solution to remove theCr (III) and any other ions which may have been present, partiallyrestored the sample peak as shown in FIG. 8.

Example 5 describes an sequence similar to Example 4, except thatnon-polar, wide pore silica separation media was used and washed withEDTA solution prior to sample injection. Once again, a sharp sample peakwas obtained, as shown in FIG. 9, when the sample was injected onto acolumn containing cleaned separation media. Deliberate contamination ofthe separation media with Cr(III) resulted in complete loss of thesample peak, as shown in FIG. 10. Injection of EDTA solution to removeCr(III), or other multivalent cations, followed by injection of the 500base pair DNA sample, partially restored the sample peak as shown inFIG. 11.

The deleterious effect of multivalent metal cations on thechromatographic separation of a 20 mer single stranded DNA standard isdescribed in Example 6 and shown by the complete loss of resolution asseen in FIG. 13 (after deliberate Cr (III) contamination) compared toFIG. 12 (column cleaned with EDTA).

The deleterious effect of even trace levels of multivalent cations ondemanding chromatographic separation is described in Example 7 and shownin FIGS. 14 and 15. A standard 4 component mixture of double strandedDNA consisting of two 209 base pair homoduplex fragments and two 209base pair heteroduplex fragments were chromatographed at 56° C. asdescribed in Example 6 on a freshly packed column DNASep column(Transgenomic, Inc., San Jose, Calif.). FIG. 14 shows only partialresolution of the 4 component mixture. However, when the column wastreated with EDTA solution followed by re-injection and elution of the 4component homoduplex/heteroduplex DNA mixture as described in Example 6,a clean separation of all 4 components was achieved, as seen in FIG. 15.This result clearly indicates that trace levels of multivalent cationswere present in a freshly packed column, and that said cationsinterfered with the separation of double stranded DNA fragments.

The method of the invention can also be used to separate polynucleotidemixtures in a batch process useful for production and isolation of purepolynucleotide fragments of a plurality of selected sizes, on a small orlarge scale. The method of the invention comprises contacting a solutionof a mixture of polynucleotide fragments containing a counterion agentwith a multivalent metal cation capture resin, followed by applying saidsolution to non-polar, wide pore separation media. The separation mediaare held in a container. The container may be a column, a membrane, acontainer, or a web. The polynucleotide mixture is held on theseparation media since the concentration of the organic component of thesolvent in which the mixture is dissolved is not sufficient to releasethe polynucleotide fragments therefrom. The separation media are thencontacted with a first eluting solvent and a counterion agent, saidfirst eluting solvent having a concentration of the organic componentsufficient to remove all polynucleotide fragments from the separationmedia which are smaller than the selected size. The eluting solvent isthen separated from the separation media. The separation media arerinsed with the first eluting solvent to remove any remaining releasedpolynucleotides. The separation media are then contacted with a secondeluting solvent and counterion agent, said second eluting solvent havinga concentration of the organic component sufficient to release thepolynucleotide fragment having the selected size from the separationmedia. The second eluting solvent is separated from the separation mediaand the particles are rinsed with the second eluting solvent. Thisprocess can be repeated to release polynucleotide of any selected sizewhich are present in the mixture.

Specific eluting solvent compositions required to elute polynucleotidefragments of any specific base pair length can be determinedexperimentally. For example, isolation of a 102 base pair fragment fromthe polynucleotide mixture may be desired, and said fragment may beeluted with 15.9% acetonitrile-water-0.1M triethylammonium acetate. Inthis example, the separation media holding the polynucleotide mixturemay be contacted with 14.6% acetonitrile-water-0.1M triethylammoniumacetate to remove reaction mixture reagents and additives, as well asall fragments having less than 102 base pairs. Increasing theacetonitrile concentration to 15.9% followed by contact of this elutingsolvent with the non-polar, wide pore separation media will release thedesired 102 base pair fragment, leaving larger fragments still attachedto the separation media. The desired polynucleotide fragment dissolvedin the eluting solvent is isolated by separating the eluting solventfrom the separation media by filtering, decanting, centrifuging, or anyother compatible liquid/solid separation technique. By using a stepgradient of increasing acetonitrile concentration, larger particles maybe removed in discreet base pair lengths from the separation media andisolated by repeating the procedure described hereinabove.

In a most preferred embodiment of the batch process of the invention,all the methods and procedures used to remove traces of multivalentcations from solvents and surfaces which contact process solution areidentical to the methods and procedures described in separation methodof the invention described hereinabove. All of process solution contractsurfaces are of materials which do not release multivalent cations. Saidmaterials are identical to those described in the separation method ofthe invention hereinabove.

The methods used to capture multivalent cations and prevent theirpresence in the batch process described hereinabove, are essential inorder to achieve high resolution separations of polynucleotides,especially double stranded DNA, and also to greatly extend the usefullife of the separation media.

The concepts, materials, systems and methods related to chromatographicseparations on non-polar, wide pore separation media are well known andare described in detail in the following references: ChromatographyToday, by Colin F. Poole and Salwa K. Pool, Elsevier (1991);Introduction to Modern Liquid Chromatography, L. R. Snyder and J. J.Kirland, J. Wiley and Sons, Inc. (1979). These references and referencescontained therein are incorporated in their entirety herein.

Non-polar, wide pore separation media and their use for the separationof polynucleotide mixtures are well known in the art and arecommercially available, e.g., Hamilton HPLC Application Handbook,(1993), Hamilton Company, Inc., 4970 Energy Way, Reno, Nev. 89502. This,and references contained therein, are incorporated in their entiretyherein. Another reference, which is incorporated in its entirety herein,describing polynucleotide separations on non-polar, wide pore reversephase particles is Chromatography, 5th edition, Part B, edited by E.Heftmann, Elsevier (1992). Separation of tRNA and DNA fragment mixtureson non-polar, wide pore silica particles is described by R. Bischoff andL.W. McLaughlin, Analytical Biochemistry, 155, 526-533 (1985) and S.Eriksson, et. al., J. Chromatography, 359, 265-274 (1986). Regardless ofthe source or composition of the non-polar, wide pore separation media,precautions are taken to ensure that they are free of multivalent cationcontaminants. For example, the separation media are washed with acidfollowed by methanol to ensure removal of residual multivalent cationcontaminants.

Procedures described in the past tense in the examples below have beencarried out in the laboratory. Procedures described in the present tensehave not been carried out in the laboratory, and are constructivelyreduced to practice with the filing of this application.

EXAMPLE 1

Acid Wash Treatment To Remove Multivalent Metal Cation Contaminants

The non-polar, wide pore reverse phase separation media were washedthree times with tetrahydrofuran, then two times with methanol. Thenon-polar, wide pore separation media were then stirred for 12 hourswith a mixture containing 100 mL of tetrahydrofuran and 100 mL ofconcentrated hydrochloric acid. Following this acid treatment, thenon-polar, wide pore separation media were washed withtetrahydrofuran/water (1:1) until neutral (pH 7). The non-polar, widepore separation media were then dried at 40° C. for 12 hours.

EXAMPLE 2

Standard Procedure for Demonstrating the Effects of Colloidal Iron onNon-polar, Wide pore Polymer Separation Media

Non-polar, wide pore PRX-1 separation media (Sarasep, Inc. San Jose,Calif.) of polystyrene/divinylbenzene polymer having a pore size of50-200 Angstroms (average pore size is 80 Angstroms) and a bead diameterof 5 microns are washed as described in Example 1 and packed in a 4.6×50mm HPLC column. A sample (5 μL, 20 ng) of 80 base pair DNA standardsolution from purified pUC18 DNA Hae III restriction enzyme digest(Sigma-Aldrich, D6293) is injected onto the column. The chromatographyis conducted under the following conditions: Eluting solvent A: 0.1 MTriethylammonium acetate (TEAA), pH 7.2; Eluting solvent B: 0.1 M TEAA,25% acetonitrile; Gradient:

    ______________________________________                                        Time (min)       % A    % B                                                   ______________________________________                                        0.0              65     35                                                    3.0              45     55                                                    10.0             35     65                                                    14.0             0      100                                                   16.0             65     35                                                    ______________________________________                                    

The flow rate is 0.75 mL/min, UV detection at 260 nm, column temp. 51°C. A peak for the 80 base pair DNA fragment is obtained. Some columns,depending on the packing volume and packing polarity, may require longertime for elution of some changes in the driving solvent concentration.

A 0.05M aqueous solution of Fe(Cl)₃ is prepared and allowed to stand atambient temperature for four hours. A 100 μL sample of the resultingcolloidal iron suspension is injected onto the column and allowed tostand for five minutes. Subsequent injection Of 5 μL of the abovedescribed 80 base pair DNA standard solution followed by the identicalgradient elution conditions described above, shows a complete absence ofany peak.

EXAMPLE 3

Standard Procedure for Demonstrating the Effects of Colloidal Iron onNon-polar, Wide pore Silica Separation Media

INERTSIL (MetaChem, Torrance, Calif.), a 5 μm C-18 non-polar, wide poreseparation medium having 200 Angstrom pores was packed in a 4.6×50 mmHPLC column and cleaned with 5 injections of 0.1 M Na₄ EDTA. A 80 basepair DNA standard (5 μL) is injected and eluted as described in Example2. A peak for the 80 base pair DNA standard is obtained.

An injection of the colloidal iron suspension is made as described inExample 3. Subsequent injection of the 80 base pair DNA standard andelution as described in Example 3 shows a complete absence of any peak.

EXAMPLE 4

Standard Procedure for Demonstrating the Effects of Chromium(III) On TheSeparation of Double Stranded DNA Using Non-polar, Wide Pore PolymerSeparation Media

Non-polar, wide pore PRX-1 separation media (Sarasep, Inc., San Jose,Calif.) of polystyrene/divinylbenzene polymer having a 5 micron diameterand a pore size of 50-200 Angstroms (80 Angstrom average pore size) waswashed as described in Example 1 and packed in 4.6×50 mm HPLC column. Asample (5 μL, 20 ng) of 500 base pair DNA standard solution fromGeneAmp^(R) Lambda Control Reagent, N808-0008, (Perkin Elmer, FosterCity, CA) was injected onto the column. The chromatography was conductedunder the following conditions: eluting solvent A; 0.1M triethylammoniumacetate (TEAA), pH 7.2; eluting solvent B; 0.1M TEAA, 25% acetonitrilegradient:

    ______________________________________                                        Time (min)       % A    % B                                                   ______________________________________                                        0.0              60     40                                                    14               0      100                                                   17               60     40                                                    ______________________________________                                    

The flow rate was 0.60 mL/min, UV detection at 260 nm, columntemperature 50° C. A peak for the 500 base pair DNA fragment wasobtained, as shown in FIG. 6.

A 520 ppm aqueous solution of Cr(III) was prepared from Cr₃ (SO₄)₂. 12H₂O and injected onto the column. The 17 min. solvent gradient was flowedthrough the column. Injection of 5 μL of the 500 base pair DNA standardonto the column and elution as described above, showed a completeabsence of any peak (FIG. 7).

A subsequent column cleanup with 3 injections, 10 μL each, of 0.1M Na₄EDTA was followed by equilibration to a constant baseline. Are-injection of 5 μL of the 500 base pair DNA standard showed areappearance of the a peak (FIG. 8) having an area of about 50% of theoriginal injection (FIG. 6).

EXAMPLE 5

Standard Procedure for Demonstrating the Effects of Chromium(III)) OnThe Separation of Double Stranded DNA Using Non-polar, Wide Pore SilicaSeparation Media

INERTSIL (MetaChem, Torrance, Calif.) 5 micron C-18 non-polar, wide pore(150 Angstrom pores) separation media was packed into a 4.6×50 mm HPLCcolumn and cleaned with 5 injections of 0.1M Na₄ EDTA. A 500 base pairDNA standard was injected onto the column and eluted as described inExample. 4. A peak for the 500 bas pair standard is shown in FIG. 9.

Injection of the Cr(III) solution as described in Example 4 followed byelution as described in Example 4, showed a complete absence of a peakas shown in FIG. 10.

Three 10 mL injections of Na₄ EDTA as described in Example 4 follwed byreinjection of the 500 base pair DNA standard and elution as describedin Example 4, showed a peak (FIG. 11 )corresponding to the 500 base pairDNA standard.

EXAMPLE 6

The Effect of Chromium(III) on the Separation of Single Stranded DNAUsing Non-polar, Wide Pore Silica Separation Media

The column described in Example 5 was cleaned with Na₄ EDTA as describedin Example 5 and equilibrated with 60%A eluting solvent to a constantbse line. A 20 mer, single stranded DNA standard (Seq2A purchased fromCTGen, Milpitas, Calif.) was injected onto the column and eluted withthe gradient protocol of Example 4. FIG. 12 shows a major peakcorresponding to the 20 mer standard and some well resolved impuritypeaks.

A single 5 mL injection of the Cr(III) solution described in Example 4was followed by re-injection of the 20 mer standard and elution usingthe gradient protocol of Example 4. The results seen in FIG. 13 show agreatly diminished peak corresponding to the 20 mer, and essentially noresolution of the impurities.

This example clearly show that metal contamination has a negative effecton the chromatography of single stranded DNA but not to the same extentas it has on double stranded DNA.

An additional injection of 5 μL onto the column followed by anotherinjection of the 20 mer standard as described above, did result incomplete elimination of the 20 mer peak. However, after 10 injections of0.1M Na₄ EDTA to remove metal contamination, as described above,followed by another injection of the 20 mer standard, did restore thepeak corresponding to the 20 mer standard. However, the peak shape wasdistorted and broad.

EXAMPLE 7

The Effect of Metal Contamination on the Separation of Heteroduplexesand Homoduplexes

A freshly packed DNASep column (Transgenomic, Inc., San Jose, Calif.)was equilibrated with eluting solvent 50% A. A mixture of DNA 209 basepair standard fragments (Transgenomic, Inc., San Jose, Calif., Cat. No.560012) consisting of 2 heteroduplex DNA fragments and 2 homoduplex DNAfragments was injected (5 μL) onto the column. The mixture was eluted ata flow rate of 0.9 mL/min using the following gradient program:

    ______________________________________                                        Time (min)       % A    % B                                                   ______________________________________                                        0.0              50     5                                                     05               47     53                                                    4.0              40     60                                                    5.5              0      100                                                   6.5              50     50                                                    8.5              50     50                                                    ______________________________________                                    

When the chromatography was run at 50° C. a normal size based separationwas observed, i.e., a single peak of unresolved hetroduplex andhomoduplex 209 base pair fragments was observed.

FIG. 14 shows the results of a chromatography run at 56° C., on a newlypacked column. Three peaks are seen. The low retention time paircorresponds to the two heteroduplexes. The large higher retention timepeak corresponds to the unresolved homoduplex peaks. The lack ofcomplete separation of all four fragments indicated that the column,though freshly packed, contained multivalent metal cation contaminants.

Column cleanup with 5, 10 μL injections of 0.1M Na₄ EDTA was followed bywashing with 25% actonitrile, 5% acetic acid, and equilibration toconstant base line with eluting solvent 50% A. Injection of the 209fragment standard homoduplex/heteroduplex mixture and elution with thegradient protocol described above, gave complete separation of themixture into four peaks, i.e., two homoduplex and 2 heteroduplex peaksas depicted in FIG. 15.

The results described in this example indicate that multivalent metalcation contamination is more critical at higher column temperatures,especially as related to the difficult separation of homoduplex andheteroduplex fragments of identical base pair length.

What is claimed is:
 1. A batch process for separating polynucleotidefragments having a selected size from a mixture of polynucleotidefragments including fragments of said selected size comprisinga)applying a solution of said polynucleotide fragments and a counterionagent to non-polar separation media having a non-polar surface, whereinsaid separation media have a pore size greater than 30 Angstroms and anaverage diameter of 1-100 microns; b) contacting the separation mediawith a first eluting solvent and counterion agent, the first elutingsolvent having a concentration of organic component sufficient torelease from the separation media all polynucleotide fragments having asize smaller than the selected size and removing the first elutingsolvent from the separation media; and c) contacting the separationmedia with a second eluting solvent having a concentration of organiccomponent sufficient to release from the separation media thepolynucleotide fragments having the selected size and removing thesecond eluting solvent from the separation media; wherein surfaces whichare contacted by the solution of polynucleotide fragments and theeluting solvent are material which does not trap or release multivalentmetal cations therefrom.
 2. A batch process of claim 1 wherein theseparation media are rinsed with fresh first eluting solvent followingstep b) to remove residual released polynucleotide fragments therefrom.3. A batch process of claim 1 wherein the separation media are rinsedwith fresh second eluting solvent following step c) to remove residualreleased polynucleotide fragments of selected size therefrom.
 4. A batchprocess of claim 1 wherein the polynucleotide mixture is double strandedor single stranded.
 5. A batch process of claim 1 wherein the solutionof polynucleotide mixture and eluting solvent have been contacted with amultivalent cation capture resin before contacting the separation media.6. A batch process of claim 5 wherein said separation media have beentreated to remove residual traces of multivalent cations therefrom.
 7. Abatch process of claim 6 wherein the solution of polynucleotide mixtureand eluting solvent have been contacted with a multivalent cationcapture resin before contacting the separation media.
 8. A batch processof claim 7 wherein the separation media are contained in a column, aweb, a membrane, or container.
 9. A batch process of claim 8 whereinsaid separation media are organic polymer or inorganic substratesselected from the group consisting of inorganic substrates, silica,zirconium, and alumina.
 10. A batch process of claim 9 wherein thenon-polar surface is an organic polymer supported on the inorganicsubstrate.
 11. A batch process of claim 9 wherein the non-polar surfaceincludes long chain hydrocarbons having from 8-24 carbons bonded theinorganic substrate.
 12. A batch process of claim 11 wherein anyresidual polar groups of the inorganic substrate have been end cappedwith trimethylsilyl chloride or hexamethyidisilazane.
 13. A batchprocess of claim 8, wherein the surfaces contacted by the solution ofpolynucleotide fragments and eluting solvent are comprised of materialselected from the group consisting of titanium, coated stainless steel,and organic polymer, or combinations thereof.
 14. A batch process ofclaim 13 wherein traces of residual multivalent metal cations have beenremoved from said surfaces by treating said surfaces with a solutioncomprising aqueous acid and chelating agent.
 15. A batch process ofclaim 13 wherein organic contaminants have been removed from saidsurfaces.
 16. A batch process of claim 7 wherein said solution ofpolynucleotide mixture and eluting solvent contain a chelating agentwhereby any trace of multivalent metal cations in the solution arecaptured.
 17. A batch process of claim 1 wherein the eluting solvent hasbeen treated to remove oxygen therefrom.